Regulation of MHC class I genes expression is critical to achieve proper immune surveillance. The majority of characterized regulatory elements are located upstream of the transcription start site. The role of downstream elements has not been investigated previously. We have now identified elements downstream of the MHC class I promoter that are necessary for appropriate regulation. Surprisingly, sequences beyond the poly A addition site are necessary for stable expression in vivo, but have no effect in transient transfection assays. Using transgenic mice and stably transfected cell lines, we have demonstrated that the 3prime DNA segment functions as a barrier element, protecting the MHC class I gene from silencing. Accordingly, truncation of the 3prime sequences results in gene silencing, increased nucleosomes density and decreased histone H3K9/K14 acetylation across the gene. Taken together, our findings demonstrate the existence of a novel barrier element downstream of the class I polyA addition site that functions to insulate the gene from silencing through epigenetic modifications. Importantly, distinct barrier element sequences are necessary to prevent silencing and to maintain an open chromatin organization across the MHC class I gene both in transgenic mice and in stably transfected cells.